The long term objective for this project is to understand the regulatory mechanisms and specific mediators of innate antiviral responses. A major contribution to host immune responses to virus infection is the production of and response to type I interferon (IFN). Virus infection or accumulated virus replication intermediates like double-stranded RNA (dsRNA) can trigger IFN biosynthesis in most cells irrespective of the presence of Toll-like receptor systems. Detection of cytosolic dsRNA is mediated by a family of sensor proteins that includes RIG-I, MDA5, and LGP2, three proteins that contain DEXD/H-box RNA helicase domains. RIG-I and MDA5 helicase domains are fused to an N-terminal region homologous to CARD domain proteins, and are positive signaling proteins leading to IFN biosynthesis. LGP2 lacks the CARD domain, and has been characterized as a feedback inhibitor for IFN synthesis. Despite the RNA helicase domain similarities, little is known about the specific enzymatic activities and how catalytic activity can influence the signaling. Preliminary results and published reports reveal a high degree of specificity in RNA recognition and differential requirements for enzymatic activity among the helicase proteins. Further specificity is revealed by natural inhibitors, paramyxovirus V proteins, that selectively target MDA5 and LGP2, but not RIG-I. This proposal will determine RNA target preferences and roles for enzymatic specialization that confer specificity and selectivity to helicase-mediated antiviral responses. The molecular basis for selective paramyxovirus helicase interference will be revealed and correlated with biological evaluation of the contributions of MDA5 and LGP2 to host responses. Regulatory mechanisms and cellular partners for helicase-mediated signaling will be characterized. These aims will expose specificity determinants for both positive and negative regulation of the intracellular innate antiviral response. Paramyxoviruses like measles, mumps human parainfluenza, and respiratory syncytial viruses, as well as other RNA viruses including influenza, HIV, Ebola and hepatitis C viruses are infectious threats to human health worldwide. The proposed experiments will reveal basic mechanisms of cellular antiviral responses and determine the molecular basis for both cellular and viral inhibition mechanisms. This strategy will reveal the strengths and vulnerabilities of the antiviral response and identify targets for therapeutic intervention.